3-substituted piperidines comprising urea functionality, and methods of use thereof

ABSTRACT

One aspect of the present invention relates to novel heterocyclic compounds comprising urea functionality. A second aspect of the present invention relates to the use of the novel heterocyclic compounds comprising urea functionality as ligands for various cellular receptors, including opioid receptors, other G-protein-coupled receptors and ion channels. An additional aspect of the present invention relates to the use of the novel heterocyclic compounds comprising urea functionality as analgesics.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/189,349, filed Mar. 14, 2000.

BACKGROUND OF THE INVENTION

Pain is an unpleasant sensation varying in severity in a local part ofthe body or several parts of the body resulting from injury, disease, oremotional disorder. Pain can be classified according to its duration.Acute pain, which lasts less than one month, usually has a readilyidentifiable cause and signals tissue damage. In addition, acute painsyndromes can be episodic, for example recurrent discomfort fromarthritis. Chronic pain can be defined as pain that persists more thanone month beyond the usual course of an acute illness or injury, or painthat recurs at intervals over months or years, or pain that isassociated with a chronic pathologic process. In contrast to acute pain,chronic pain loses its adaptive biologic function. Depression is common,and abnormal illness behavior often compounds the patient's impairment.

Millions of people suffer from chronic or intractable pain. Persistentpain varies in etiology and presentation. In some cases, symptoms andsigns may be evident within a few weeks to a few months after theoccurrence of an injury or the onset of disease, e.g. cancer or AIDS.Like many illnesses that at one time were not well understood, pain andits many manifestations may be poorly treated and seriouslyunderestimated. Inappropriately treated pain seriously compromises thepatient's quality of life, causing emotional suffering and increasingthe risk of lost livelihood and disrupted social integration. Severechronic pain affects both the pediatric and adult population, and oftenleads to mood disorders, including depression and, in rare cases,suicide.

In the last several years, health policy-makers, health professionals,regulators, and the public have become increasingly interested in theprovision of better pain therapies. This interest is evidenced, in part,by the U.S. Department of Health and Human Services' dissemination ofClinical Practice Guidelines for the management of acute pain and cancerpain. There is currently no nationally accepted consensus for thetreatment of chronic pain not due to cancer, yet the economic and socialcosts of chronic pain are substantial, with estimates ranging in thetens of billions of dollars annually.

Three general classes of drugs are currently available for painmanagement, nonsteriodal anti-inflammatories, opioids, and adjuvantanalgesics. The nonsteriodal anti-inflammatories class includes drugssuch as aspirin, ibuprofen, diclofenac, acetaminophen, celecoxib, androfecoxib. The opioid class includes morphine, oxycodone, fentanyl, andpentazocine. Adjuvant analgesics include various antidepressants,anticonvulsants, neuroleptics, and corticosteroids.

Opioids are the major class of analgesics used in the management ofmoderate to severe pain because of their effectiveness, ease oftitration, and favorable risk-to-benefit ratio. Opioids produceanalgesia by binding to specific receptors both within and outside theCNS. Opioid analgesics are classified as full agonists, partialagonists, or mixed agonist-antagonists, depending on the receptors towhich they bind and their intrinsic activities at each receptor.

Three subclasses of opioid receptor have been identified in humans,namely the δ-. κ-, and μ-opioid receptors. Analgesia is thought toinvolve activation of μ and/or κ receptors. Notwithstanding their lowselectivity for μ over κ receptors, it is likely that morphine andmorphine-like opioid agonists produce analgesia primarily throughinteraction with μ receptors; selective agonists of κ receptors inhumans produce analgesia, because rather than the euphoria associatedwith morphine and congeners, these compounds often produce dysphoria andpsychotomimetic effects. The consequences of activating δ receptors inhumans remain unclear.

Although opioids can be very effective in pain management, they do causeseveral side effects, such as respiratory depression, constipation,physical dependence, tolerance, withdraw. These unwanted effects canseverely limit their use.

Opioids are known to produce respiratory depression that is proportionalto their analgesia. This respiratory depression can be life threatening.This results in a narrow range between the effective dose and a dosethat produces respiratory depression. Because of this narrow therapeuticindex, patients receiving opioid therapy must be closely monitored forsigns of respiratory failure.

Opioids can also cause constipation in individuals receiving them. Thisside effect can be severe and may require prolonged hospitalization, oreven surgical intervention.

Commonly used full agonists include morphine, hydromorphone, meperidine,methadone, levorphanol, and fentanyl. These opioids are classified asfull agonists because there is not a ceiling to their analgesicefficacy, nor will they reverse or antagonize the effects of otheropioids within this class when given simultaneously. Side effectsinclude respiratory depression, constipation, nausea, urinary retention,confusion, and sedation. Morphine is the most commonly used opioid formoderate to severe pain because of its availability in a wide variety ofdosage forms, its well-characterized pharmacokinetics andpharmacodynamics, and its relatively low cost. Meperidine may be usefulfor brief courses (e.g., a few days) to treat acute pain and to managerigors (shivering) induced by medication, but it generally should beavoided in patients with cancer because of its short duration of action(2.5 to 3.5 hours) and its toxic metabolite, normeperidine. Thismetabolite accumulates, particularly when renal function is impaired,and causes CNS stimulation, which may lead to dysphoria, agitation, andseizures; meperidine, therefore, should not be used if continued opioiduse is anticipated.

The development of physical dependence with repeated use is acharacteristic feature of the opioid drugs, and the possibility ofdeveloping drug dependence is one of the major limitations of theirclinical use. Almost all opioid users rapidly develop drug dependencywhich can lead to apathy, weight loss, loss of sex drive, anxiety,insomnia, and drug cravings. Although physical dependence is common,addiction and abuse are not common in pain patients who are treatedappropriately with opioid drugs.

Historically, the development of analgesic tolerance was believed tolimit the ability to use opioids efficaciously on a long-term basis forpain management. Tolerance, or decreasing pain relief with the samedosage over time, has not proven to be a prevalent limitation tolong-term opioid use. Experience with treating cancer pain has shownthat what initially appears to be tolerance is usually progression ofthe disease. Furthermore, for most opioids, there does not appear to bean arbitrary upper dosage limit, as was once thought.

Cessation of opioid administration may result in a withdrawal syndrome.Symptoms of withdrawal are often the opposite of the effects achieved bythe drug; withdrawal from morphine, however, results in complex symptomsthat may seem unrelated to its effects. Misunderstanding of addictionand mislabeling of patients as addicts result in unnecessary withholdingof opioid medications. Addiction is a compulsive disorder in which anindividual becomes preoccupied with obtaining and using a substance, thecontinued use of which results in a decreased quality of life. Studiesindicate that the de novo development of addiction is low when opioidsare used for the relief of pain. Furthermore, even opioid addicts canbenefit from the carefully supervised, judicious use of opioids for thetreatment of pain due to cancer, surgery, or recurrent painful illnessessuch as sickle cell disease.

The known opioids have been very effective in pain management. However,they have restricted use because of several potentially severe sideeffects. Therefore, there is a current need for pharmaceutical agentsthat retain the analgesic properties of the known opioid, but that havereduced side effect profiles.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to novel heterocycliccompounds comprising urea functionality. A second aspect of the presentinvention relates to the use of the novel heterocyclic compoundscomprising urea functionality as ligands for various cellular receptors,including opioid receptors, other G-protein-coupled receptors and ionchannels. An additional aspect of the present invention relates to theuse of the novel heterocyclic compounds comprising urea functionality asanalgesics.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the IC₅₀s of four compounds of the present inventionagainst site 2 of sodium channels, and type L calcium channels.

FIG. 2 presents biological data for five compounds of the presentinvention in various assays focused on opioid receptors.

DETAILED DESCRIPTION OF THE INVENTION

Pain is an unpleasant sensation varying in severity in a local part ofthe body or several parts of the body resulting from injury, disease, oremotional disorder. Pain can be classified according to its duration.Acute pain, which lasts less than one month, usually has a readilyidentifiable cause (e.g., hip fracture) and signals tissue damage. Theassociated effect is often anxiety, and the concomitant physiologicfindings are those of sympathetic stimulation (e.g., tachycardia,tachypnea, diaphoresis). In addition, acute pain syndromes can beepisodic, for example recurrent discomfort from arthritis.

Chronic pain can be defined as pain that persists more than one monthbeyond the usual course of an acute illness or injury, or pain thatrecurs at intervals over months or years, or pain that is associatedwith a chronic pathologic process. In contrast to acute pain, chronicpain loses its adaptive biologic function. Depression is common, andabnormal illness behavior often compounds the patient's impairment.Chronic pain can be divided broadly into that which is inferred to bepredominantly somatogenic and that which is inferred to be predominantlypsychogenic. A similar classification based on inferred pathophysiologydesignates chronic pain as nociceptive (commensurate with ongoingactivation of pain-sensitive nerve fibers), neuropathic (due to aberrantsomatosensory processing in afferent neural pathways), or psychogenic.

Nociceptive pain can be somatic or visceral. Most chronic pain in theelderly is nociceptive and somatic; arthritis, cancer pain, andmyofascial pain are most common. Relief is likely with removal of theperipheral cause (e.g., reducing periarticular inflammation), andanalgesic drugs are often effective.

A common subtype of neuropathic pain, known collectively as peripheralneuropathic pain, is presumably sustained by mechanisms that involvedisturbances in the peripheral nerve or nerve root; neuroma formationafter axonal injury and nerve compression are the two major processes.Another subtype of neuropathic pain is related to the reorganization ofnociceptive information processing by the CNS; it persists withoutongoing activation of pain-sensitive fibers. This type of pain, knowncollectively as the deafferentation syndromes, includes postherpeticneuralgia, central pain (which can result from a lesion at any level ofthe CNS), phantom limb pain, and others. A third subtype of neuropathicpain, often called sympathetically maintained pain, can be amelioratedby interruption of sympathetic nerves to the painful area; theprototypic disorder is reflex sympathetic dystrophy. The precisemechanisms involved in these disorders are conjectural, but all canproduce an unfamiliar pain, often described as burning and stabbing.Currently, this type of pain responds poorly to analgesics.

Some patients have persistent pain without either nociceptive foci orevidence of a neuropathic mechanism for the pain. Many others havenociceptive lesions that do not sufficiently explain the degree of painand disability. Psychopathologic processes account for these complaintsin some patients. If no evidence for a psychological cause is found, thepain is referred to as idiopathic. Many patients have an idiopathic painsyndrome that is best described by the generic diagnosis chronicnonmalignant pain syndrome, a term denoting pain and disabilitydisproportionate to an identifiable somatic cause and usually related toa more pervasive set of abnormal illness behaviors. Some of thesepatients may be labeled by the more formal psychiatric diagnosis ofsomatoform pain disorder. Others have complaints that constitute aspecific pain diagnosis, most commonly the failed low back syndrome oratypical facial pain. Still others have significant organic lesions(e.g., lumbar arachnoiditis) but also have a clear psychologicalcontribution associated with excessive disability. Diagnosis may bedifficult, but the relative contributions of both organic andpsychological components of the pain can be defined.

Another clinically useful classification of chronic pain is broadlysyndromic. For example, chronic pain may be part of a medical illness(e.g., cancer or arthritis). A mixture of pathophysiologic mechanismsmay be involved; e.g., tumor invasion of nerve and bone may causeneuropathic and somatic nociceptive pains, respectively, andpsychological factors may be prominent.

Three general classes of drugs are currently available for painmanagement, nonsteriodal anti-inflammatories, opioids, and adjuvantanalgesics. The nonsteriodal anti-inflammatories class includes drugssuch as aspirin, ibuprofen, diclofenac, acetaminophen, and rofecoxib.The opioid class includes morphine, oxycodone, fentanyl, andpentazocine. Adjuvant analgesics include various antidepressants,anticonvulsants, neuroleptics, and corticosteroids.

Of the three classes of pharmaceutical agents used for pain management,opioid are usually most efficacious for treating moderate to severepain. Although opioids can be very effective in pain management, they docause several side effects, such as respiratory depression,constipation, physical dependence, tolerance, withdraw. These unwantedeffects can severely limit their use. Therefore, there is a current needfor pharmaceutical agents that retain the analgesic properties of theknown opioid, but have reduced side effect profiles for the treatment ofpain.

Opioids, specifically ligands for the μ-opioid receptor, are the majorclass of analgesics used in the management of moderate to severe painbecause of their effectiveness, ease of titration, and favorablerisk-to-benefit ratio. Unfortunately, the opioids currently availablehave several unwanted side-effects, such as respiratory depression andconstipation. In addition, these agents may lead to tolerance anddependence. Research into the development of new, selective ligands foropioid receptors holds the promise of yielding potent analgesics thatlack the side effects of morphine and its congeners. Applicants hereindisclose novel analgesics, including selective ligands for opioidreceptors. Individual compounds described herein promise to haveagonistic, antagonistic, and hybrid effects on opioid and other cellularreceptors. Additionally, new compounds reported herein may possessanalgesic properties free from respiratory depression and the potentialfor physical dependence associated with μ-opioid receptor ligands, suchas morphine and fentanyl. Moreover, new compounds reported herein maypossess properties for the treatment of physical or psychologicaladditions, psychiatric disorders, and neurological pathologies, such astinnitus.

The μ-opioid receptor is a member of a family of cell surface proteinsthat permit intracellular transduction of extracellular signals. Cellsurface proteins provide eukaryotic and prokaryotic cells a means todetect extracellular signals and transduce such signals intracellularlyin a manner that ultimately results in a cellular response or aconcerted tissue or organ response. Cell surface proteins, byintracellularly transmitting information regarding the extracellularenvironment via specific intracellular pathways induce an appropriateresponse to a particular stimulus. The response may be immediate andtransient, slow and sustained, or some mixture thereof. By virtue of anarray of varied membrane surface proteins, eukaryotic cells areexquisitely sensitive to their environment.

Extracellular signal molecules, such as growth hormones, vasodilatorsand neurotransmitters, exert their effects, at least in part, viainteraction with cell surface proteins. For example, some extracellularsignal molecules cause changes in transcription of target gene viachanges in the levels of secondary messengers, such as cAMP. Othersignals, indirectly alter gene expression by activating the expressionof genes, such as immediate-early genes that encode regulatory proteins,which in turn activate expression of other genes that encodetranscriptional regulatory proteins. For example, neuron gene expressionis modulated by numerous extracellular signals, includingneurotransmitters and membrane electrical activity. Transsynapticsignals cause rapid responses in neurons that occur over a period oftime ranging from milleseconds, such as the opening of ligandgatedchannels, to seconds and minutes, such as second messenger-mediatedevents. Genes in neural cells that are responsive to transsynapticstimulation and membrane electrical activity, include genes, calledimmediate early genes, whose transcription is activated rapidly, withinminutes, and transiently (see, e.g., Sheng et al. (1990) Neuron 4:477-485), and genes whose expression requires protein synthesis andwhose expression is induced or altered over the course of hours.

Cell surface receptors and ion channels are among the cell surfaceproteins that respond to extracellular signals and initiate the eventsthat lead to this varied gene expression and response. Ion channels andcell surface-localized receptors are ubiquitous and physiologicallyimportant cell surface membrane proteins. They play a central role inregulating intracellular levels of various ions and chemicals, many ofwhich are important for cell viability and function.

Cell surface-localized receptors are membrane spanning proteins thatbind extracellular signalling molecules or changes in the extracellularenvironment and transmit the signal via signal transduction pathways toeffect a cellular response. Cell surface receptors bind circulatingsignal polypeptides, such as neurotransmitters, growth factors andhormones, as the initiating step in the induction of numerousintracellular pathways. Receptors are classified on the basis of theparticular type of pathway that is induced. Included among these classesof receptors are those that bind growth factors and have intrinsictyrosine kinase activity, such as the heparin binding growth factor(HBGF) receptors, and those that couple to effector proteins throughguanine nucleotide binding regulatory proteins, which are referred to asG protein coupled receptors and G proteins, respectively.

The G protein transmembrane signaling pathways consist of threeproteins: receptors, G proteins and effectors. G proteins, which are theintermediaries in transmembrane signaling pathways, are heterodimers andconsist of alpha , beta and gamma subunits. Among the members of afamily of G proteins the alpha subunits differ. Functions of G proteinsare regulated by the cyclic association of GTP with the alpha subunitfollowed by hydrolysis of GTP to GDP and dissociation of GDP.

G protein coupled receptors are a diverse class of receptors thatmediate signal transduction by binding to G proteins. Signaltransduction is initiated via ligand binding to the cell membranereceptor, which stimulates binding of the receptor to the G protein. Thereceptor G protein interaction releases GDP, which is specifically boundto the G protein, and permits the binding of GTP, which activates the Gprotein. Activated G protein dissociates from the receptor and activatesthe effector protein, which regulates the intracellular levels ofspecific second messengers. Examples of such effector proteins includeadenyl cyclase, guanyl cyclase, phospholipase C, and others.

G protein-coupled receptors, which are glycoproteins, are known to sharecertain structural similarities and homologies (see, e.g., Gilman, A.G., Ann. Rev. Biochem. 56: 615-649 (1987), Strader, C. D. et al. TheFASEB Journal 3: 1825-1832 (1989), Kobilka, B. K., et al. Nature329:75-79 (1985) and Young et al. Cell 45: 711-719 (1986)). Among the Gprotein-coupled receptors that have been identified and cloned are thesubstance P receptor, the angiotensin receptor, the alpha- andbeta-adrenergic receptors and the serotonin receptors. G protein-coupledreceptors share a conserved structural motif. The general and commonstructural features of the G protein-coupled receptors are the existenceof seven hydrophobic stretches of about 20-25 amino acids eachsurrounded by eight hydrophilic regions of variable length. It has beenpostulated that each of the seven hydrophobic regions forms atransmembrane alpha helix and the intervening hydrophilic regions formalternately intracellularly and extracellularly exposed loops. The thirdcytosolic loop between transmembrane domains five and six is theintracellular domain responsible for the interaction with G proteins.

G protein-coupled receptors are known to be inducible. This inducibilitywas originally described in lower eukaryotes. For example, the cAMPreceptor of the cellular slime mold, Dictyostelium, is induced duringdifferentiation (Klein et al., Science 241: 1467-1472 (1988). During theDictyostelium discoideum differentiation pathway, cAMP, induces highlevel expression of its G protein-coupled receptor. This receptortransduces the signal to induce the expression of the other genesinvolved in chemotaxis, which permits multicellular aggregates to align,organize and form stalks (see, Firtel, R. A., et al. Cell 58: 235-239(1989) and Devreotes, P., Science 245: 1054-1058 (1989)).

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “cell surface proteins” includes molecules that occur on thesurface of cells, interact with the extracellular environment, andtransmit or transduce information regarding the environmentintracellularly.

The term “extracellular signals” includes a molecule or a change in theenvironment that is transduced intracellularly via cell surface proteinsthat interact, directly or indirectly, with the signal. An extracellularsignal is any compound or substance that in some manner specificallyalters the activity of a cell surface protein. Examples of such signalsinclude, but are not limited to, molecules such as acetylcholine, growthfactors, hormones and other mitogenic substances, such as phorbolmistric acetate (PMA), that bind to cell surface receptors and ionchannels and modulate the activity of such receptors and channels.Extracellular signals also includes as yet unidentified substances thatmodulate the activity of a cell surface protein and thereby affectintracellular functions and that are potential pharmacological agentsthat may be used to treat specific diseases by modulating the activityof specific cell surface receptors.

The term “ED₅₀” means the dose of a drug which produces 50% of itsmaximum response or effect. Alternatively, the dose which produces apredetermined response in 50% of test subjects or preparations.

The term “LD₅₀” means the dose of a drug which is lethal in 50% of testsubjects.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

The term “structure-activity relationship (SAR)” refers to the way inwhich altering the molecular structure of drugs alters their interactionwith a receptor, enzyme, etc.

The term “agonist” refers to a compound that mimics the action ofnatural transmitter or, when the natural transmitter is not known,causes changes at the receptor complex in the absence of other receptorligands.

The term “antagonist” refers to a compound that binds to a receptorsite, but does not cause any physiological changes unless anotherreceptor ligand is present.

The term “competitive antagonist” refers to a compound that binds to areceptor site; its effects can be overcome by increased concentration ofthe agonist.

The term “partial agonist” refers to a compound that binds to a receptorsite but does not produce the maximal effect regardless of itsconcentration.

The term “ligand” refers to a compound that binds at the receptor site.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (σ)constant. This well known constant is described in many references, forinstance, J. March, Advanced Organic Chemistry, McGraw Hill BookCompany, New York, (1977 edition) pp. 251-259. The Hammett constantvalues are generally negative for electron donating groups (σ[P]=−0.66for NH₂) and positive for electron withdrawing groups (σ[P]=0.78 for anitro group), σ[P] indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, acyl, formyl, sulfonyl,trifluoromethyl, cyano, chloride, and the like. Exemplaryelectron-donating groups include amino, methoxy, and the like.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a group permittedby the rules of valence.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)-R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S-(CH₂)_(m)-R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)-R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)-R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′¹¹ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O-(CH₂)_(m)-R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonylamino” is art recognized and includes a moiety thatcan be represented by the general formula:

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

The term “sulfonyl”, as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkylalkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term “sulfoxido” as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se-(CH₂)_(m)-R₇, m and R₇ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., functioning as analgesics), whereinone or more simple variations of substituents are made which do notadversely affect the efficacy of the compound in binding to opioidreceptors. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Compounds of the Invention

In certain embodiments, the compounds of the present invention arerepresented by general structure A:

wherein

X represents CH₂, NR, O or S;

Z represents O, S or NR;

R represents independently for each occurrence H, alkyl, aryl orheteroaryl; and two geminal instances of R taken together may representO;

R represents H, alkyl alkenyl, alkynyl, aryl, heteroaryl, aralkyl orheteroaralkyl;

R₂ represents independently for each occurrence H, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl or heteroaralkyl, or the twoinstances of R₂ taken together represent an aryl or heteroaryl moiety ora —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, or —CH₂CH₂CH₂CH₂-tether between the twonitrogens of the urea moiety;

R₃ represents H, alkyl, fluoroalkyl, aryl, alkenyl, alkynyl, heteroaryl,aralkyl or heteroaralkyl;

m is an integer in the range 1 to 4 inclusive;

n is an integer in the range 0 to 2 inclusive;

p is an integer in the range 0 to 3 inclusive; and

the stereochemical configuration at any stereocenter of a compoundrepresented by A may be R, S, or a mixture of these configurations.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein Z represents O.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein R represents independently for each occurrence H or alkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein n is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂; and Z represents O.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂; and R represents independently for eachoccurrence H or alkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂; and n is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein Z represents O; and R represents independently for eachoccurrence H or alkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein Z represents O; and n is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein Z represents O; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein n is 1; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂; Z represents O; and R represents independentlyfor each occurrence H or alkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂; Z represents O; R represents independently foreach occurrence H or alkyl; and n is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂; Z represents O; R represents independently foreach occurrence H or alkyl; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents CH₂; Z represents O; R represents independently foreach occurrence H or alkyl; n is 1; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions, or anyof the narrower definitions, wherein said compound is a singleenantiomer.

In assays based on mammalian G-protein-coupled receptors, opioidreceptors or ion channels, certain compounds according to generalstructure A have IC₅₀ values less than 10 μM, more preferably less than5 μM, and most preferably less than 1 μM.

In assays based on opioid receptors from mammalian brain, certaincompounds according to general structure A have IC₅₀, values less than10 μM against at least one subclass of opioid receptor, more preferablyless than 5 μM, and most preferably less than 1 μM.

In certain embodiments, the compounds of the present invention arerepresented by general structure B:

X represents CH₂, NR, O or S;

Z represents O, S or NR;

R represents independently for each occurrence H, alkyl, aryl orheteroaryl; and two geminal instances of R taken together may representO;

R₁ represents H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl orheteroaralkyl;

R₂ represents H, alkyl, fluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl or heteroaralkyl;

R₃ is absent or present 1, 2, 3 or 4 times;

R₃ represents independently for each occurrence alkyl, aryl, alkenyl,alkynyl, heteroaryl, aralkyl, heteroaralkyl, halogen, —N(R)₂, formyl,acyl, —CO₂R, —CONR₂, —O₂CR, acylamino, —SR, or —OR;

m is an integer in the range 1 to 4 inclusive;

n is an integer in the range 0 to 2 inclusive;

p is an integer in the range 0 to 3 inclusive; and

the stereochemical configuration at any stereocenter of a compoundrepresented by B may be R, S, or a mixture of these configurations.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein Z represents O.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein R represents independently for each occurrence H or alkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein R₃ is absent.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein n is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; and Z represents O.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; and R represents independently for eachoccurrence H or alkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; and R₃ is absent.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; and n is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein Z represents O; and R represents independently for eachoccurrence H or alkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein Z represents O; and R₃ is absent.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein Z represents O; and n is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein Z represents O; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein n is 1; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; Z represents O; and R represents independentlyfor each occurrence H or alkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; Z represents O; R represents independently foreach occurrence H or alkyl; and R₃ is absent.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; Z represents O; R represents independently foreach occurrence H or alkyl; and n is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; Z represents O; R represents independently foreach occurrence H or alkyl; and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; Z represents O; R represents independently foreach occurrence H or alkyl; R₃ is absent; n is 1;and p is 0 or 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents CH₂; Z represents O; R represents independently foreach occurrence H; R₁ is phenyl; R₂ represents H; R₃ is absent; m is 2;n is 1; and p is 1.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions, or anyof the narrower definitions, wherein said compound is a singleenantiomer.

In assays based on mammalian G-protein-coupled receptors, opioidreceptors or ion channels, certain compounds according to generalstructure B have IC₅₀ values less than 10 μM, more preferably less than5 μM, and most preferably less than 1 μM.

In assays based on opioid receptors from mammalian brain, certaincompounds according to general structure B have IC₅₀ values less than 10μM against at least one subclass of opioid receptor, more preferablyless than 5 μM, and most preferably less than 1 μM.

In certain embodiments, the present invention relates to ligands forG-protein-coupled receptors, opioid receptors or ion channels, whereinthe ligands are represented by any of generalized structures A or B, andany of the sets of definitions associated with one of those structures.Preferably the ligands of the present invention are antagonists oragonists of the G-protein-coupled receptors, opioid receptors or ionchannels. In any event, the ligands of the present invention preferablyexert their effect on the receptors or channels at a concentration lessthan about 10 micromolar, more preferably at a concentration less thanabout 1 micromolar, and most preferably at a concentration less than 100nanomolar. In certain embodiments, the ligands of the present inventionbind selectively to a single family of G-protein-coupled receptors,opioid receptors or ion channels. In other embodiments, the ligands ofthe present invention bind selectively to a subtype of receptor orchannel within a family of G-protein-coupled receptors, opioid receptorsor ion channels.

In certain embodiments, the selectivity of a ligand for a specificfamily or subtype of receptor or channel renders that ligand aneffective therapeutic agent for an acute or chronic ailment, disease ormalady. In certain embodiments, the selectivity of a ligand for aspecific family or subtype of receptor or channel consists of a bindingaffinity for that family or subtype of receptor or channel at least afactor of ten greater than its binding affinity for other families orsubtypes of G-protein-coupled receptors, opioid receptors or ionchannels. In preferred embodiments, the selectivity of a ligand for aspecific family or subtype of receptor or channel consists of a bindingaffinity for that family or subtype of receptor or channel at least afactor of one hundred greater than its binding affinity for otherfamilies or subtypes of G-protein-coupled receptors, opioid receptors orion channels. In certain embodiments, the selectivity of a ligand for aspecific family or subtype of receptor or channel consists of a bindingaffinity for that family or subtype of receptor or channel at least afactor of one thousand greater than its binding affinity for otherfamilies or subtypes of G-protein-coupled receptors, opioid receptors orion channels.

The present invention contemplates pharmaceutical formulations (seebelow) of the compounds of the present invention. In certainembodiments, the pharmaceutical formulations will comprise compounds ofthe present invention that effect only a specific family or subtype ofG-protein-coupled receptors, opioid receptors or ion channels, andthereby have a therapeutic effect on an acute or chronic ailment,disease or malady that is at least in part due to biochemical orphysiological processes associated with the receptor(s) or channel(s).In preferred embodiments, the pharmaceutical formulations will comprisecompounds of the present invention that effect only a subtype ofreceptor or channel, and thereby have a therapeutic effect on an acuteor chronic ailment, disease or malady that is at least in part due tobiochemical or physiological processes associated with the specificsubtype of receptor or channel. The Background of the Invention (seeabove) teaches examples of acute or chronic ailments, diseases ormaladies that are caused or exacerbated by biochemical or physiologicalprocesses associated with certain G-protein-coupled receptors, opioidreceptors or ion channels. One of ordinary skill in the art will be ableto accumulate, by reference to the scientific literature, a morecomprehensive list of acute or chronic ailments, diseases or maladiesthat are caused or exacerbated by biochemical or physiological processesassociated with specific G-protein-coupled receptors, opioid receptorsor ion channels. The present invention contemplates pharmaceuticalformulations of compounds of the present invention that will be ofmedicinal value against the aforementioned acute or chronic ailments,diseases or maladies.

Biochemical Activity at Cellular Receptors, and Assays to Detect ThatActivity

Assaying processes are well known in the art in which a reagent is addedto a sample, and measurements of the sample and reagent are made toidentify sample attributes stimulated by the reagent. For example, onesuch assay process concerns determining in a chromogenic assay theamount of an enzyme present in a biological sample or solution. Suchassays are based on the development of a colored product in the reactionsolution. The reaction develops as the enzyme catalyzes the conversionof a colorless chromogenic substrate to a colored product.

Assays useful in the present invention concern determining the activityof receptors the activation of which initiates subsequent intracellularevents in which intracellular stores of calcium ions are released foruse as a second messenger. Activation of some G-protein-coupledreceptors stimulates the formation of inositol triphosphate (IP3, aG-protein-coupled receptor second messenger) through phospholipaseC-mediated hydrolysis of phosphatidylinositol, Berridge and Irvine(1984). Nature 312:315-21. IP3 in turn stimulates the release ofintracellular calcium ion stores.

A change in cytoplasmic calcium ion levels caused by release of calciumions from intracellular stores is used to determine G-protein-coupledreceptor function. This is another type of indirect assay. AmongG-protein-coupled receptors are muscarinic acetylcholine receptors(mAChR), adrenergic receptors, serotonin receptors, dopamine receptors,angiotensin receptors, adenosine receptors, bradykinin receptors,metabotropic excitatory amino acid receptors and the like. Cellsexpressing such G-protein-coupled receptors may exhibit increasedcytoplasmic calcium levels as a result of contribution from bothintracellular stores and via activation of ion channels, in which caseit may be desirable although not necessary to conduct such assays incalcium-free buffer, optionally supplemented with a chelating agent suchas EGTA, to distinguish fluorescence response resulting from calciumrelease from internal stores. Another type of indirect assay involvesdetermining the activity of receptors which, when activated, result in achange in the level of intracellular cyclic nucleotides, e.g., cAMP,cGMP. For example, activation of some dopamine, serotonin, metabotropicglutamate receptors and muscarinic acetylcholine receptors results in adecrease in the cAMP or cGMP levels of the cytoplasm.

Furthermore, there are cyclic nucleotide-gated ion channels, e.g., rodphotoreceptor cell channels and olfactory neuron channels [see,Altenhofen, W. et al. (1991) Proc. Natl. Acad. Sci U.S.A. 88:9868-9872and Dhallan et al. (1990) Nature 347:184-187] that are permeable tocations upon activation by binding of cAMP or cGMP. A change incytoplasmic ion levels caused by a change in the amount of cyclicnucleotide activation of photo-receptor or olfactory neuron channels isused to determine function of receptors that cause a change in cAMP orcGMP levels when activated. In cases where activation of the receptorresults in a decrease in cyclic nucleotide levels, it may be preferableto expose the cells to agents that increase intracellular cyclicnucleotide levels, e.g., forskolin, prior to adding areceptor-activating compound to the cells in the assay. Cell for thistype of assay can be made by co-transfection of a host cell with DNAencoding a cyclic nucleotide-gated ion channel and a DNA encoding areceptor (e.g., certain metabotropic glutamate receptors, muscarinicacetylcholine receptors, dopamine receptors, serotonin receptors and thelike, which, when activated, causes a change in cyclic nucleotide levelsin the cytoplasm.

Any cell expressing a receptor protein which is capable, uponactivation, of directly increasing the intracellular concentration ofcalcium, such as by opening gated calcium channels, or indirectlyaffecting the concentration of intracellular calcium as by causinginitiation of a reaction which utilizes Ca<2+> as a second messenger(e.g., G-protein-coupled receptors), may form the basis of an assay.Cells endogenously expressing such receptors or ion channels and cellswhich may be transfected with a suitable vector encoding one or moresuch cell surface proteins are known to those of skill in the art or maybe identified by those of skill in the art. Although essentially anycell which expresses endogenous ion channel and/or receptor activity maybe used, it is preferred to use cells transformed or transfected withheterologous DNAs encoding such ion channels and/or receptors so as toexpress predominantly a single type of ion channel or receptor. Manycells that may be genetically engineered to express a heterologous cellsurface protein are known. Such cells include, but are not limited to,baby hamster kidney (BHK) cells (ATCC No. CCL10), mouse L cells (ATCCNo. CCLI.3), DG44 cells [see, Chasin (1986) Cell. Molec. Genet. 12:555]human embryonic kidney (HEK) cells (ATCC No. CRL1573), Chinese hamsterovary (CHO) cells (ATCC Nos. CRL9618, CCL61, CRL9096), PC12 cells (ATCCNo. CRL1721) and COS-7 cells (ATCC No. CRL1651). Preferred cells forheterologous cell surface protein expression are those that can bereadily and efficiently transfected. Preferred cells include HEK 293cells, such as those described in U.S. Pat. No. 5,024,939.

Any compound which is known to activate ion channels or receptors ofinterest may be used to initiate an assay. Choosing an appropriate ionchannel- or receptor-activating reagent depending on the ion channel orreceptor of interest is within the skill of the art. Directdepolarization of the cell membrane to determine calcium channelactivity may be accomplished by adding a potassium salt solution havinga concentration of potassium ions such that the final concentration ofpotassium ions in the cell-containing well is in the range of about50-150 mM (e.g., 50 mM KCl). With respect to ligand-gated receptors andligand-gated ion channels, ligands are known which have affinity for andactivate such receptors. For example, nicotinic acetylcholine receptorsare known to be activated by nicotine or acetylcholine; similarly,muscarinic acetylcholine receptors may be activated by addition ofmuscarine or carbamylcholine.

Agonist assays may be carried out on cells known to possess ion channelsand/or receptors to determine what effect, if any, a compound has onactivation or potentiation of ion channels or receptors of interest.Agonist assays also may be carried out using a reagent known to possession channel- or receptor-activating capacity to determine whether a cellexpresses the respective functional ion channel or receptor of interest.

Contacting a functional receptor or ion channel with agonist typicallyactivates a transient reaction; and prolonged exposure to an agonist maydesensitize the receptor or ion channel to subsequent activation. Thus,in general, assays for determining ion channel or receptor functionshould be initiated by addition of agonist (i.e., in a reagent solutionused to initiate the reaction). The potency of a compound having agonistactivity is determined by the detected change in some observable in thecells (typically an increase, although activation of certain receptorscauses a decrease) as compared to the level of the observable in eitherthe same cell, or substantially identical cell, which is treatedsubstantially identically except that reagent lacking the agonist (i.e.,control) is added to the well. Where an agonist assay is performed totest whether or not a cell expresses the functional receptor or ionchannel of interest, known agonist is added to test-cell-containingwells and to wells containing control cells (substantially identicalcell that lacks the specific receptors or ion channels) and the levelsof observable are compared. Depending on the assay, cells lacking theion channel and/or receptor of interest should exhibit substantially noincrease in observable in response to the known agonist. A substantiallyidentical cell may be derived from the same cells from which recombinantcells are prepared but which have not been modified by introduction ofheterologous DNA. Alternatively, it may be a cell in which the specificreceptors or ion channels are removed. Any statistically or otherwisesignificant difference in the level of observable indicates that thetest compound has in some manner altered the activity of the specificreceptor or ion channel or that the test cell possesses the specificfunctional receptor or ion channel.

In an example of drug screening assays for identifying compounds whichhave the ability to modulate ion channels or receptors of interest,individual wells (or duplicate wells, etc.) contain a distinct celltype, or distinct recombinant cell line expressing a homogeneouspopulation of a receptor or ion channel of interest, so that thecompound having unidentified activity may be screened to determinewhether it possesses modulatory activity with respect to one or more ofa variety of functional ion channels or receptors. It is alsocontemplated that each of the individual wells may contain the same celltype so that multiple compounds (obtained from different reagent sourcesin the apparatus or contained within different wells) can be screenedand compared for modulating activity with respect to one particularreceptor or ion channel type.

Antagonist assays, including drug screening assays, may be carried outby incubating cells having functional ion channels and/or receptors inthe presence and absence of one or more compounds, added to the solutionbathing the cells in the respective wells of the microtiter plate for anamount of time sufficient (to the extent that the compound has affinityfor the ion channel and/or receptor of interest) for the compound(s) tobind to the receptors and/or ion channels, then activating the ionchannels or receptors by addition of known agonist, and measuring thelevel of observable in the cells as compared to the level of observablein either the same cell, or substantially identical cell, in the absenceof the putative antagonist.

The assays are thus useful for rapidly screening compounds to identifythose that modulate any receptor or ion channel in a cell. Inparticular, assays can be used to test functional ligand-receptor orligand-ion channel interactions for cell receptors includingligand-gated ion channels, voltage-gated ion channels, G-protein-coupledreceptors and growth factor receptors.

Those of ordinary skill in the art will recognize that assays mayencompass measuring a detectable change of a solution as a consequenceof a cellular event which allows a compound, capable of differentialcharacteristics, to change its characteristics in response to thecellular event. By selecting a particular compound which is capable ofdifferential characteristics upon the occurrence of a cellular event,various assays may be performed. For example, assays for determining thecapacity of a compound to induce cell injury or cell death may becarried out by loading the cells with a pH-sensitive fluorescentindicator such as BCECF (Molecular Probes, Inc., Eugene, Oreg. 97402,Catalog #B1150) and measuring cell injury or cell death as a function ofchanging fluorescence over time.

In a further example of useful assays, the function of receptors whoseactivation results in a change in the cyclic nucleotide levels of thecytoplasm may be directly determined in assays of cells that expresssuch receptors and that have been injected with a fluorescent compoundthat changes fluorescence upon binding cAMP. The fluorescent compoundcomprises cAMP-dependent-protein kinase in which the catalytic andregulatory subunits are each labelled with a different fluorescent-dye[Adams et al. (1991) Nature 349:694-697]. When cAMP binds to theregulatory subunits, the fluorescence emission spectrum changes; thischange can be used as an indication of a change in cAMP concentration.

The function of certain neurotransmitter transporters which are presentat the synaptic cleft at the junction between two neurons may bedetermined by the development of fluorescence in the cytoplasm of suchneurons when conjugates of an amine acid and fluorescent indicator(wherein the fluorescent indicator of the conjugate is an acetoxymethylester derivative e.g., 5-(aminoacetamido)fluorescein; Molecular Probes,Catalog #A1363) are transported by the neurotransmitter transporter intothe cytoplasm of the cell where the ester group is cleaved by esteraseactivity and the conjugate becomes fluorescent.

In practicing an assay of this type, a reporter gene construct isinserted into an eukaryotic cell to produce a recombinant cell which haspresent on its surface a cell surface protein of a specific type. Thecell surface receptor may be endogenously expressed or it may beexpressed from a heterologous gene that has been introduced into thecell. Methods for introducing heterologous DNA into eukaryotic cellsare-well known in the art and any such method may be used. In addition,DNA encoding various cell surface proteins is known to those of skill inthe art or it may be cloned by any method known to those of skill in theart.

The recombinant cell is contacted with a test compound and the level ofreporter gene expression is measured. The contacting may be effected inany vehicle and the testing may be by any means using any protocols,such as serial dilution, for assessing specific molecular interactionsknown to those of skill in the art. After contacting the recombinantcell for a sufficient time to effect any interactions, the level of geneexpression is measured. The amount of time to effect such interactionsmay be empirically determined, such as by running a time course andmeasuring the level of transcription as a function of time. The amountof transcription may be measured using any method known to those ofskill in the art to be suitable. For example, specific mRNA expressionmay be detected using Northern blots or specific protein product may beidentified by a characteristic stain. The amount of transcription isthen compared to the amount of transcription in either the same cell inthe absence of the test compound or it may be compared with the amountof transcription in a substantially identical cell that lacks thespecific receptors. A substantially identical cell may be derived fromthe same cells from which the recombinant cell was prepared but whichhad not been modified by introduction of heterologous DNA.Alternatively, it may be a cell in which the specific receptors areremoved. Any statistically or otherwise significant difference in theamount of transcription indicates that the test compound has in somemanner altered the activity of the specific receptor.

If the test compound does not appear to enhance, activate or induce theactivity of the cell surface protein, the assay may be repeated andmodified by the introduction of a step in which the recombinant cell isfirst tested for the ability of a known agonist or activator of thespecific receptor to activate transcription if the transcription isinduced, the test compound is then assayed for its ability to inhibit,block or otherwise affect the activity of the agonist.

The transcription based assay is useful for identifying compounds thatinteract with any cell surface protein whose activity ultimately altersgene expression. In particular, the assays can be used to testfunctional ligand-receptor or ligand-ion channel interactions for anumber of categories of cell surface-localized receptors, including:ligand-gated ion channels and voltage-gated ion channels, and Gprotein-coupled receptors.

Any transfectable cell that can express the desired cell surface proteinin a manner such the protein functions to intracellularly transduce anextracellular signal may be used. The cells may be selected such thatthey endogenously express the cell surface protein or may be geneticallyengineered to do so. Many such cells are known to those of skill in theart. Such cells include, but are not limited to Ltk<−> cells, PC12 cellsand COS-7 cells.

The preparation of cells which express a receptor or ion channel and areporter gene expression construct, and which are useful for testingcompounds to assess their activities, is exemplified in the Examplesprovided herewith by reference to mammalian Ltk<−> and COS-7 cell lines,which express the Type I human muscarinic (HM1) receptor and which aretransformed with either a c-fos promoter-CAT reporter gene expressionconstruct or a c-fos promoter-luciferase reporter gene expressionconstruct.

Any cell surface protein that is known to those of skill in the art orthat may be identified by those of skill in the art may used in theassay. The cell surface protein may endogenously expressed on theselected cell or it may be expressed from cloned DNA. Exemplary cellsurface proteins include, but are not limited to, cell surface receptorsand ion channels. Cell surface receptors include, but are not limitedto, muscarinic receptors (e.g.,, human M2 (GenBank accession #M16404);rat M3 (GenBank accession #M16407); human M4 (GenBank accession#M16405); human M5 (Bonner et al. (1988) Neuron 1:403-410); and thelike); neuronal nicotinic acetylcholine receptors (e.g., the alpha 2,alpha 3 and beta 2 subtypes disclosed in U.S. Ser. No. 504,455 (filedApr. 3, 1990), hereby expressly incorporated by reference herein in itsentirety); the rat alpha 2 subunit (Wada et al. (1988) Science240:330-334); the rat alpha 3 subunit (Boulter et al. (1986) Nature319:368-374); the rat alpha 4 subunit (Goldman et al. (1987) cell48:965-973); the rat alpha 5 subunit (Boulter et al. (1990) J. Biol.Chem. 265:4472-4482); the rat beta 2 subunit (Deneris et al. (1988)Neuron 1:45-54); the rat beta 3 subunit (Deneris et al. (1989) J. Biol.Chem. 264: 6268-6272); the rat beta 4 subunit (Duvoisin et al. (1989)Neuron 3:487-496); combinations of the rat alpha subunits, beta subunitsand alpha and beta subunits; GABA receptors (e.g., the bovine alpha 1and beta 1 subunits (Schofield et al. (1987) Nature 328:221-227); thebovine alpha 2 and alpha 3 subunits (Levitan et al. (1988) Nature335:76-79); the gamma-subunit (Pritchett et al. (1989) Nature338:582-585); the beta 2 and beta 3 subunits (Ymer et alo (1989) EMBO J.8:1665-1670); the delta subunit (Shivers, B. D. (1989) Neuron3:327-337); and the like); glutamate receptors (e.g., receptor isolatedfrom rat brain (Hollmann et al. (1989) Nature 342:643-648); and thelike); adrenergic receptors (e.g., human beta 1 (Frielle et al. (1987)Proc. Natl. Acad. Sci. 84.:7920-7924); human alpha 2 (Kobilka et al.(1987) Science 238:650-656); hamster beta 2 (Dixon et al. (1986) Nature321:75-79); and the like); dopamine receptors (e.g., human D2 (Stormannet al. (1990) Molec. Pharm.37:1-6); rat (Bunzow et al. (1988) Nature336:783-787); and the like); NGF receptors (e.g., human NGF receptors(Johnson et al. (1986) Cell 47:545-554); and the like); serotoninreceptors (e.g., human 5HT1a (Kobilka et al. (1987) Nature 329:75-79);rat 5HT2 (Julius et al. (1990) PNAS 87:928-932); rat 5HT1c (Julius etal. (1988) Science 241:558-564); and the like).

Reporter gene constructs are prepared by operatively linking a reportergene with at least one transcriptional regulatory element. If only onetranscriptional regulatory element is included it must be a regulatablepromoter, At least one of the selected transcriptional regulatoryelements must be indirectly or directly regulated by the activity of theselected cell-surface receptor whereby activity of the receptor can bemonitored via transcription of the reporter genes.

The construct may contain additional transcriptional regulatoryelements, such as a FIRE sequence, or other sequence, that is notnecessarily regulated by the cell surface protein, but is selected forits ability to reduce background level transcription or to amplify thetransduced signal and to thereby increase the sensitivity andreliability of the assay.

Many reporter genes and transcriptional regulatory elements are known tothose of skill in the art and others may be identified or synthesized bymethods known to those of skill in the art.

A reporter gene includes any gene that expresses a detectable geneproduct, which may be RNA or protein. Preferred reporter genes are thosethat are readily detectable. The reporter gene may also be included inthe construct in the form of a fusion gene with a gene that includesdesired transcriptional regulatory sequences or exhibits other desirableproperties.

Examples of reporter genes include, but are not limited to CAT(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature282: 864-869) luciferase, and other enzyme detection systems, such asbeta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell.Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984),PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667);alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238,Hall et al. (1983) J. Mol. Appl. Gen. 2: 101).

Transcriptional control elements include, but are not limited to,promoters, enhancers, and repressor and activator binding sites,Suitable transcriptional regulatory elements may be derived from thetranscriptional regulatory regions of genes whose expression is rapidlyinduced, generally within minutes, of contact between the cell surfaceprotein and the effector protein that modulates the activity of the cellsurface protein. Examples of such genes include, but are not limited to,the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477-485),such as c-fos, Immediate early genes are genes that are rapidly inducedupon binding of a ligand to a cell surface protein. The transcriptionalcontrol elements that are preferred for use in the gene constructsinclude transcriptional control elements from immediate early genes,elements derived from other genes that exhibit some or all of thecharacteristics of the immediate early genes, or synthetic elements thatare constructed such that genes in operative linkage therewith exhibitsuch characteristics. The characteristics of preferred genes from whichthe transcriptional control elements are derived include, but are notlimited to, low or undetectable expression in quiescent cells, rapidinduction at the transcriptional level within minutes of extracellularsimulation, induction that is transient and independent of new proteinsynthesis, subsequent shut-off of transcription requires new proteinsynthesis, and mRNAs transcribed from these genes have a shorthalf-life. It is not necessary for all of these properties to bepresent.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; or (8) nasally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred per cent, this amount will range fromabout 1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and a compound of thepresent invention. In certain embodiments, an aforementioned formulationrenders orally bioavailable a compound of the present invention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; (8) absorbents, such as kaolinand bentonite clay; (9) lubricants, such a talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-shelled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or oral cavity; or (4)intravaginally or intravectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compounds of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”, W.H. Freedman and Co., San Francisco, U.S.A., 1969 or “Livestock Feeds andFeeding” O and B books, Corvallis, Ore., U.S.A., 1977).

Combinatorial Libraries

The subject reactions readily lend themselves to the creation ofcombinatorial libraries of compounds for the screening ofpharmaceutical, agrochemical or other biological or medically-relatedactivity or material-related qualities. A combinatorial library for thepurposes of the present invention is a mixture of chemically relatedcompounds which may be screened together for a desired property; saidlibraries may be in solution or covalently linked to a solid support.The preparation of many related compounds in a single reaction greatlyreduces and simplifies the number of screening processes which need tobe carried out. Screening for the appropriate biological,pharmaceutical, agrochemical or physical property may be done byconventional methods.

Diversity in a library can be created at a variety of different levels.For instance, the substrate aryl groups used in a combinatorial approachcan be diverse in terms of the core aryl moiety, e.g., a variegation interms of the ring structure, and/or can be varied with respect to theother substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules. See, for example,Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat.Nos. 5,359,115 and 5,362,899: the Ellman U.S. Pat. No. 5,288,514: theStill et al. PCT publication WO 94/08051; Chen et al. (1994) JACS116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092,WO93/09668 and WO91/07087; and the Lerner et al. PCT publicationWO93/20242). Accordingly, a variety of libraries on the order of about16 to 1,000,000 or more diversomers can be synthesized and screened fora particular activity or property.

In an exemplary embodiment, a library of substituted diversomers can besynthesized using the subject reactions adapted to the techniquesdescribed in the Still et al. PCT publication WO 94/08051, e.g., beinglinked to a polymer bead by a hydrolyzable or photolyzable group, e.g.,located at one of the positions of substrate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. In one embodiment, which is particularly suitable for discoveringenzyme inhibitors, the beads can be dispersed on the surface of apermeable membrane, and the diversomers released from the beads by lysisof the bead linker. The diversomer from each bead will diffuse acrossthe membrane to an assay zone, where it will interact with an enzymeassay. Detailed descriptions of a number of combinatorial methodologiesare provided below.

A) Direct Characterization

A growing trend in the field of combinatorial chemistry is to exploitthe sensitivity of techniques such as mass spectrometry (MS), e.g.,which can be used to characterize sub-femtomolar amounts of a compound,and to directly determine the chemical constitution of a compoundselected from a combinatorial library. For instance, where the libraryis provided on an insoluble support matrix, discrete populations ofcompounds can be first released from the support and characterized byMS. In other embodiments, as part of the MS sample preparationtechnique, such MS techniques as MALDI can be used to release a compoundfrom the matrix, particularly where a labile bond is used originally totether the compound to the matrix. For instance, a bead selected from alibrary can be irradiated in a MALDI step in order to release thediversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis

The libraries of the subject method can take the multipin libraryformat. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS81:3998-4002) introduced a method for generating compound libraries by aparallel synthesis on polyacrylic acid-grated polyethylene pins arrayedin the microtitre plate format. The Geysen technique can be used tosynthesize and screen thousands of compounds per week using the multipinmethod, and the tethered compounds may be reused in many assays.Appropriate linker moieties can also been appended to the pins so thatthe compounds may be cleaved from the supports after synthesis forassessment of purity and further evaluation (c.f., Bray et al. (1990)Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166).

C) Divide-Couple-Recombine

In yet another embodiment, a variegated library of compounds can beprovided on a set of beads utilizing the strategy ofdivide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135;and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971). Briefly, as thename implies, at each synthesis step where degeneracy is introduced intothe library, the beads are divided into separate groups equal to thenumber of different substituents to be added at a particular position inthe library, the different substituents coupled in separate reactions,and the beads recombined into one pool for the next iteration.

In one embodiment, the divide-couple-recombine strategy can be carriedout using an analogous approach to the so-called “tea bag” method firstdeveloped by Houghten, where compound synthesis occurs on resin sealedinside porous polypropylene bags (Houghten et al. (1986) PNAS82:5131-5135). Substituents are coupled to the compound-bearing resinsby placing the bags in appropriate reaction solutions, while all commonsteps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

D) Combinatorial Libraries by Light-Directed, Spatially AddressableParallel Chemical Synthesis A scheme of combinatorial synthesis in whichthe identity of a compound is given by its locations on a synthesissubstrate is termed a spatially-addressable synthesis. In oneembodiment, the combinatorial process is carried out by controlling theaddition of a chemical reagent to specific locations on a solid support(Dower et al. (1991) Annu Rep Med Chem 26:271-280; Fodor, S. P. A.(1991) Science 251:767; Pirrung et al. (1992) U.S. Pat. No. 5,143,854;Jacobs et al. (1994) Trends Biotechnol 12:19-26). The spatial resolutionof photolithography affords miniaturization. This technique can becarried out through the use protection/deprotection reactions withphotolabile protecting groups.

The key points of this technology are illustrated in Gallop et al.(1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared forcoupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers or otherphotolabile linkers. Light is used to selectively activate a specifiedregion of the synthesis support for coupling. Removal of the photolabileprotecting groups by light (deprotection) results in activation ofselected areas. After activation, the first of a set of amino acidanalogs, each bearing a photolabile protecting group on the aminoterminus, is exposed to the entire surface. Coupling only occurs inregions that were addressed by light in the preceding step. The reactionis stopped, the plates washed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized simultaneously; this characteristic leads to thegeneration of many different masking strategies.

E) Encoded Combinatorial Libraries

In yet another embodiment, the subject method utilizes a compoundlibrary provided with an encoded tagging system. A recent improvement inthe identification of active compounds from combinatorial librariesemploys chemical indexing systems using tags that uniquely encode thereaction steps a given bead has undergone and, by inference, thestructure it carries. Conceptually, this approach mimics phage displaylibraries, where activity derives from expressed peptides, but thestructures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. A variety of other forms of encodinghave been reported, including encoding with sequenceable bio-oligomers(e.g., oligonucleotides and peptides), and binary encoding withadditional non-sequenceable tags.

1) Tagging with Sequenceable Bio-Oligomers

The principle of using oligonucleotides to encode combinatorialsynthetic libraries was described in 1992 (Brenner et al. (1992) PNAS89:5381-5383), and an example of such a library appeared the followingyear (Needles et al. (1993) PNAS 90:10700-10704). A combinatoriallibrary of nominally 7⁷ (=823,543) peptides composed of all combinationsof Arg, Gln, Phe, Lys, Val, D-Val and Thr (three-letter amino acidcode), each of which was encoded by a specific dinucleotide (TA, TC, CT,AT, TT, CA and AC, respectively), was prepared by a series ofalternating rounds of peptide and oligonucleotide synthesis on solidsupport. In this work, the amine linking functionality on the bead wasspecifically differentiated toward peptide or oligonucleotide synthesisby simultaneously preincubating the beads with reagents that generateprotected OH groups for oligonucleotide synthesis and protected NH₂groups for peptide synthesis (here, in a ratio of 1:20). When complete,the tags each consisted of 69-mers, 14 units of which carried the code.The bead-bound library was incubated with a fluorescently labeledantibody, and beads containing bound antibody that fluoresced stronglywere harvested by fluorescence-activated cell sorting (FACS). The DNAtags were amplified by PCR and sequenced, and the predicted peptideswere synthesized. Following such techniques, compound libraries can bederived for use in the subject method, where the oligonucleotidesequence of the tag identifies the sequential combinatorial reactionsthat a particular bead underwent, and therefore provides the identity ofthe compound on the bead.

The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesisof non-oligomeric libraries. In preferred embodiments, the librariesemploy linkers permitting selective detachment of the test compoundlibrary member for assay.

Peptides have also been employed as tagging molecules for combinatoriallibraries. Two exemplary approaches are described in the art, both ofwhich employ branched linkers to solid phase upon which coding andligand strands are alternately elaborated. In the first approach (KerrJ. M. et al. (1993) J Am Chem Soc 115:2529-2531), orthogonality insynthesis is achieved by employing acid-labile protection for the codingstrand and base-labile protection for the compound strand.

In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170),branched linkers are employed so that the coding unit and the testcompound can both be attached to the same functional group on the resin.In one embodiment, a cleavable linker can be placed between the branchpoint and the bead so that cleavage releases a molecule containing bothcode and the compound (Ptek et al. (1991) Tetrahedron Lett32:3891-3894). In another embodiment, the cleavable linker can be placedso that the test compound can be selectively separated from the bead,leaving the code behind. This last construct is particularly valuablebecause it permits screening of the test compound without potentialinterference of the coding groups. Examples in the art of independentcleavage and sequencing of peptide library members and theircorresponding tags has confirmed that the tags can accurately predictthe peptide structure.

2) Non-Sequenceable Tagging: Binary Encoding

An alternative form of encoding the test compound library employs a setof non-sequencable electrophoric tagging molecules that are used as abinary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926). Exemplary tagsare haloaromatic alkyl ethers that are detectable as theirtrimethylsilyl ethers at less than femtomolar levels by electron capturegas chromatography (ECGC). Variations in the length of the alkyl chain,as well as the nature and position of the aromatic halide substituents,permit the synthesis of at least 40 such tags, which in principle canencode 2⁴⁰ (e.g., upwards of 10¹²) different molecules. In the originalreport (Ohlmeyer et al., supra) the tags were bound to about 1% of theavailable amine groups of a peptide library via a photocleavableo-nitrobenzyl linker. This approach is convenient when preparingcombinatorial libraries of peptide-like or other amine-containingmolecules. A more versatile system has, however, been developed thatpermits encoding of essentially any combinatorial library. Here, thecompound would be attached to the solid support via the photocleavablelinker and the tag is attached through a catechol ether linker viacarbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem59:4723-4724). This orthogonal attachment strategy permits the selectivedetachment of library members for assay in solution and subsequentdecoding by ECGC after oxidative detachment of the tag sets.

Although several amide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the electrophoric tags are attacheddirectly to the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031)and provide guidance for generating the subject compound library. Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second,compounds are partially detached and transferred to assay plates; third,a metal binding assay identifies the active wells; fourth, thecorresponding beads are rearrayed singly into new microtiter plates;fifth, single active compounds are identified; and sixth, the structuresare decoded.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLE 1 Synthesis of3-Ethyl-1-(1-phenethyl-piperidin-3-ylmethyl)-1-phenyl Urea (3)

To a solution of N-(1-Boc-piperidin-3-ylmethyl)aniline 1 (102 mg, 0.35mmol) in 1 mL of dry CH₂Cl₂was added ethyl isocyanate (95%, 35 μL, 1.2eq.) at room temperature. After being shaken at room temperature forovernight, the reaction mixture was passed through an aminopropyl NH₂cartridge and washed with CH₂Cl₂. Removal of the CH₂Cl₂ affordedN-(1-Boc-piperidin-3-yl-methyl)-N-phenyl-N′-ethylurea (2) (120 mg, 95%).LRMS 285 (M-100)⁺ 261.

Trifluoroacetic acid (0.5 mL) was added dropwise to a solution of 2 (81mg, 0.225 mmol) in 0.5 mL of dry CH₂Cl₂ at 0° C. (ice-water). Thereaction mixture was stirred at room temperature for 30 minutes. TLCshowed the reaction was completed. After removal of the solvents, theresidue was dried under vacuum for 3 hrs. The crude product was used fornext step without purification.

The crude compound from previous step was dissolved in DMF (1.0 mL) andphenylacetaldehyde (60 mg, 2 eq.) was added. The mixture was stirred atroom temperature for 30 min. NaB(OAc)₃H (95%, 100 mg, 2 eq.) wasintroduced in one portion, and the mixture was shaken at roomtemperature overnight. The mixture was quenched with 5 mL of aqueouspotassium carbonate (sat.), then extracted with ethyl acetate (3×10 mL).The extracts were combined and washed with aqueous NaHCO₃ (sat., 2×5mL), brine (10 mL), and dried over anhydrous sodium sulfate. After thesolvent was removed, the remaining oily residue was purified by apreparative thin layer chromatography (EtOAc/MeOH, 9:1) to give3-Ethyl-1-(1-phenethyl-piperidin-3-ylmethyl)-1-phenyl-urea (3) as acolorless oil (46 mg, 56%). LRMS 365.

EXAMPLE 2 Synthesis of 1-(1-phenethylpiperidin-3-ylmethyl)-1,3-diphenylUrea (4)

Compound 4 was synthesized according to the procedure outlined inExample 1, using phenyl isocyanate in place of ethyl isocyante. 4: LRMS413.

EXAMPLE 3 Synthesis of1-(1-phenethylpiperidin-3-ylmethyl)-1-phenyl-3-(3-trifluoromethylphenyl)Urea (5)

Compound 5 was synthesized according to the procedure outlined inExample 1, using 3-trifluoromethylphenyl isocyanate in place of ethylisocyante. 5: LRMS 481.

EXAMPLE 4 Synthesis of1-(1-phenethylpiperidin-3-ylmethyl)-1-phenyl-3-(4-trifluoromethylphenyl)Urea (6)

Compound 6 was synthesized according to the procedure outlined inExample 1, using 4-trifluoromethylphenyl isocyanate in place of ethylisocyante. 6: LRMS 481.

EXAMPLE 5 Synthesis of1-(1-phenethylpiperidin-3-ylmethyl)-1-phenyl-3-(4-trifluoromethoxyphenyl)Urea (7)

Compound 7 was synthesized according to the procedure outlined inExample 1, using 4-trifluoromethoxyphenyl isocyanate in place of ethylisocyante. 7: LRMS 497.

EXAMPLE 6 Synthesis ofN-1-Carbobenzyloxy[3-(2′-Amino(anilino))carboxy]piperidine (8)

A solution of Cbz-nipecotic acid (5.81 mmol, 1.53 g) and1,2-phenylenediamine (2.0 equiv, 11.6 mmol, 1.26 g) in CH₂Cl₂ (15 mL) at0° C. was treated with DCC (1.5 equiv, 8.72 mmol, 1.80 g) under Ar. Thereaction mixture was allowed to warm to 25° C. and stirred for 12 h. Thereaction mixture was then filtered to remove the urea and the solventswere removed in vacuo. Chromatography (SiO₂, 2.5 cm×30.5 cm, 1:1hexane-EtOAc) gave a product that was still contaminated with1,2-phenylenediamine. The resulting oil was dissolved in EtOAc andhexane was slowly added until yellowish precipitate formed. Theresulting solid was filtered and washed with hexanes and dried in vacuoto give 8 (1.50 g, 2.05 g theoretical, 73%) as a yellowish white solid:R_(f) 0.13 (SiO₂, 1:1 hexane-EtOAc); LRMS m/z 353 (M⁺, C₂₀H₂₃N₃O₃,requires 353).

EXAMPLE 7 Synthesis ofN-1-Carbobenzyloxy[3-(1′-(N-(2′-ketobenzyimidazolinyl)))-methylene]piperidine(9)

A solution of 8 (0.28 mmol, 100 mg) in THF (0.5 mL) at 0° C. was treatedwith 1.0M BH₃-THF (1.5 equiv, 0.46 mmol) under Ar. The reaction mixturewas then heated to 80° C. and allowed to stir for 2.5 h. The reactionmixture was then cooled to 0° C. and quenched with 10% aqueous HCl. ThepH was adjusted to 10 with 10% aqueous NaOH and the reaction mixture wasextracted with 3×EtOAc (25 mL). The organics were washed with saturatedaqueous NaCl, and dried over MgSO₄. Based on poor stability the materialwas carried directly to the next step.

The above solution in CH₂Cl₂ (1.5 mL) at 0° C. was treated withtriphosgene (1.0 equiv, 0.28 mmol, 83 mg) and pyridine (6.0 equiv, 1.68mmol, 136 μL) under Ar. The reaction mixture warmed to 25° C. andstirred for 3 h. The reaction mixture was cooled to 0° C. and quenchedwith 10% aqueous NaHCO₃. The reaction mixture was then made acidic with10% aqueous HCl and extracted with 3×EtOAc (25 mL). The organics werewashed with saturated aqueous NaCl, dried over MgSO₄ and the solventswere removed in vacuo. Chromatography (PTLC, SiO₂, 20 cm×20 cm, 1 mm,9:1 CH₂Cl₂—CH₃OH) provided 9 (59 mg, 102 mg theoretical, 58%) as a whitesolid: R_(f) 0.22 (SiO₂, 1:1 hexane-EtOAc); LRMS m/z 365 (M⁺,C₂₁H₂₃N₃O₃, requires 353).

EXAMPLE 8 Synthesis ofN-1-Phenethyl[3-(1′-(N-(2′-ketobenzyimidazolinyl)))methylene]piperidine

A solution of 9 (0.096 mmol, 35 mg), phenylacetaldehyde (10.0 equiv,0.96 mmol, 110 μL), and 10% Pd-C (10 mg) in CH₃OH (2 mL) at 25° C. wastreated with H₂ at 48 psi. The reaction mixture was stirred for 5 h. Thereaction mixture was filtered through Celite and the solvents wereremoved in vacuo. Chromatography (PTLC, SiO₂, 20 cm×20 cm, 1 mm, 9:1EtOAc-CH₃OH) provided 10 (13 mg, 32 mg theoretical, 41%) as a whitesolid: R_(f) 0.25 (SiO₂, 9:1 EtOAc-CH₃OH); ¹H NMR (CDCl₃, 300 MHz) 89.75 (s, 1H), 7.36-7.00 (m, 9H), 3.83-3.80 (d, 2H), 2.98-2.76 (m, 4H),2.66-2.54 (m, 2H), 2.38-2.22 (m, 1H), 2.20-1.97 (m, 2H), 1.84-1.54 (m,4H); ¹³C NMR (CDCl₃, 75 MHz) δ 156.0, 140.7, 130.9, 129.0, 128.6, 128.1,126.2, 121.7, 121.5, 109.8, 108.5, 61.2, 58.1, 54.1, 44.8, 36.1, 30.0,28.7, 24.9; LRMS m/z 335 (M⁺, C₂₁H₂₅N₃O, requires 335).

EXAMPLE 9 Synthesis ofN-1-tert-Butoxycarbonyl[3-R-(2′-Amino(anilino))carboxy]piperidine (11)

A solution of Boc-R-nipecotic acid (18.8 mmol, 4.00 g) and1,2-phenylenediamine (1.0 equiv, 18.8 mmol, 1.26 g) in CH₂Cl₂ (35 mL) at0° C. was treated with DCC (1.5 equiv, 28.2 mmol, 5.82 g) under Ar. Thereaction mixture was allowed to warm to 25° C. and stirred for 12 h. Thereaction mixture was then filtered to remove the urea and the solventswere removed in vacuo. Chromatography (SiO₂, 2.5 cm×30.5 cm, 2:1hexane-EtOAc) provided 11 (3.58 g, 6.00 g theoretical, 60%) as ayellowish white solid; LRMS m/z 319 (M⁺, C₁₇H₂₅N₃O₃, requires 319).

EXAMPLE 10 Synthesis ofN-1-tert-Butoxycarbonyl[3-R-(1′-(N-(2′-ketobenzyimidazolinyl)))methylene]piperidine(12)

A solution of 11 (11.2 mmol, 3.58 g) in THF (15 mL) at 0° C. was treatedwith 1.0M BH₃-THF (2.0 equiv, 22 mmol) under Ar. The reaction mixturewas then heated to 80° C. and allowed to stir for 12 h. The reactionmixture was then cooled to 0° C. and quenched with 10% aqueous HCl. ThepH was adjusted to 10 with 10% aqueous NaOH and the reaction mixture wasextracted with EtOAc (3×25 mL). The organics were washed with saturatedaqueous NaCl, and dried over MgSO₄. Based on poor stability the materialwas carried directly to the next step.

The above compound in THF (50 mL) at 0° C. was treated with phosgene(20% in toluene) (2.0 equiv, 21.5 mmol) and triethylamine (2.0 equiv,21.5 mmol, 3.00 mL) under Ar. The reaction mixture warmed to 25° C. andstirred for 3 h. The reaction mixture was cooled to 0° C. and quenchedwith 10% aqueous NaHCO₃. The reaction mixture was then made acidic with10% aqueous HCl and extracted with EtOAc (3×25 mL). The organics werewashed with saturated aqueous NaCl, dried over MgSO₄, and the solventswere removed in vacuo. Chromatography (SiO₂, 2.5 cm×30.5 cm,3:2EtOAc-hexane) provided 12 (2.16 g, 3.56 mg theoretical, 61%) as awhite solid; LRMS m/z 331 (M⁺, C₁₈H₂₅N₃O₃, requires 331).

EXAMPLE 11N-1-Phenethyl[3-R-(1′-(N-(2′-ketobenzyimidazolinyl)))methylene]piperidine(13)

A solution of 12 (6.52 mmol, 2.16 g) in CH₂Cl₂ (15 mL) at 25° C. wastreated with 50% TFA in CH₂Cl₂ (15 mL). The reaction mixture was stirredfor 2 h. The solvents were removed in vacuo and the resulting oil wastreated with phenethyl bromide (2.0 equiv, 13.0 mmol, 1.78 mL) and K₂CO₃(4.0 equiv, 26.1 mmol, 3.60 g) in CH₃CN (30 mL). The reaction mixturestirred for 12 h at 65° C. and then quenched with 30 mL of H₂O. Thereaction mixture was then extracted with EtOAc (30 mL). The organiclayer was washed with saturated aqueous NaCl, and dried over MgSO₄.Chromatography (SiO₂, 2.5 cm×30.5 cm, 9:1 EtOAc-CH₃OH) provided 13 (1.91g, 2.19 g theoretical, 87%) as a white solid; LRMS m/z 335 (M⁺,C₂₁H₂₅N₃O, requires 335).

EXAMPLE 12N-1-tert-Butoxycarbonyl[3-S-(2′-Amino(anilino))carboxy]piperidine (14)

A solution of Boc-S-nipecotic acid (14.1 mmol, 3.00 g) and1,2-phenylenediamine (1.0 equiv, 14.1 mmol, 1.52 g) in CH₂Cl₂ (35 mL) at0° C. was treated with DCC (1.5 equiv, 21.2 mmol, 4.37 g) under Ar. Thereaction mixture was allowed to warm to 25° C. and stirred for 12 h. Thereaction mixture was then filtered to remove the urea and the solventswere removed in vacuo. Chromatography (SiO₂, 2.5 cm×30.5 cm, 2:1hexane-EtOAc) gave 14 (2.99 g, 4.50 g theoretical, 66%) as a yellowishwhite solid; LRMS m/z 319 (M⁺, C₂OH₂₃N₃O₃, requires 319).

EXAMPLE 13 N-1-tert-Butoxycarbonyl[3-S-(1′-(N-(2′-ketobenzyimidazolinyl)))methylene]piperidine (15)

A solution of 14 (9.36 mmol, 2.99 g) in THF (10 mL) at 0° C. was treatedwith 1.0M BH₃-THF (2.0 equiv, 19 mmol) under Ar. The reaction mixturewas then heated to 80° C. and allowed to stir for 12 h. The reactionmixture was then cooled to 0° C. and quenched with 10% aqueous HCl. ThepH was adjusted to 10 with 10% aqueous NaOH and the reaction mixture wasextracted with EtOAc (3×25 mL). The organics were washed with saturatedaqueous NaCl, and dried over MgSO₄. Based on poor stability the materialwas carried directly to the next step.

The above compound in THF (40 mL) at 0° C. was treated with phosgene(20% in toluene) (2.0 equiv, 16.3 mmol) and triethylamine (2.0 equiv,16.3 mmol, 2.30 mL) under Ar. The reaction mixture warmed to 25° C. andstirred for 3 h. The reaction mixture was cooled to 0° C. and quenchedwith 10% aqueous NaHCO₃. The reaction mixture was then made acidic with10% aqueous HCl and extracted with EtOAc (3×25 mL). The organics werewashed with saturated aqueous NaCl, dried over MgSO₄, and the solventswere removed in vacuo. Chromatography (SiO₂, 2.5 cm×30.5 cm, 3:2EtOAc-hexane) provided 15 (2.16 g, 3.56 mg theoretical, 61%) as a whitesolid; LRMS m/z 331 (M⁺, C₁₈H₂₅N₃O₃, requires 331).

EXAMPLE 14N-1-Phenethyl[3-S-(1′-(N-(2′-ketobenzyimidazolinyl)))methylene]piperidine(16)

A solution of 15 (5.40 mmol, 1.79 g) in CH₂Cl₂ (12 mL) at 25° C. wastreated with 50% TFA in CH₂Cl₂ (12 mL) . The reaction mixture stirredfor 2 h. The solvents were removed in vacuo and the resulting oil wastreated with phenethyl bromide (2.0 equiv, 10.8 mmol, 1.50 mL) and K₂CO₃(4.0 equiv, 21.6 mmol, 3.00 g) in CH₃CN (20 mL). The reaction mixturestirred for 12 h at 65° C. and then quenched with 30 mL of H₂O. Thereaction mixture was then extracted with EtOAc (30 mL). The organicswere washed with saturated aqueous NaCl, and dried over MgSO₄.Chromatography (SiO₂, 2.5 cm×30.5 cm, 9:1 EtOAc-CH₃OH) provided 16 (1.29g, 1.81 g theoretical, 71%) as a white solid; LRMS m/z 335 (M⁺,C₂₁H₂₅N₃O, requires 335).

EXAMPLE 15N-1-Carbobenzyloxy[3-(1′-(N-(3′-ethyl-2′-ketobenzyimidazolinyl)))methylene]-piperidine(17)

A solution of 9 (0.274 mmol, 100 mg) and ethyl bromide (1.5 equiv, 0.411mmol, 31 μL) in DMF (0.5 mL) at 25° C. was treated with NaH (60% inmineral oil) (1.5 equiv, 0.411 mmol, 17 mg). The reaction mixturestirred for 12 h at 25° C. and then quenched with 1 mL of NaHCO₃ (sat)The reaction mixture was then extracted with EtOAc (3 mL). The organiclayer was washed with saturated aqueous NaCl, and dried over MgSO₄.Chromatography (PTLC, SiO₂, 20 cm×20 cm, 1 mm, 1:1 EtOAc-hexane)provided 17 (104 mg, 108 mg theoretical, 96%) as a white solid; LRMS m/z393 (M⁺, C₂₃H₂₇N₃O₃, requires 393).

EXAMPLE 16 N-1-Phenethyl[3-(1′-(N-(3′-ethyl-2′-ketobenzyimidazolinyl)))methylene]piperidine (18)

Compound 17 (0.25 mmol, 97 mg and 10% Pd-C (10 mg) in CH₃OH (1 mL) at25° C. was treated with H₂ via a balloon and stirred for 5 h. Thereaction mixture was then filtered through Celite and the solvents wereremoved in vacuo. The resulting oil was treated with phenethyl bromide(1.5 equiv, 0.38 mmol, 52 μL) and K₂CO₃ (1.5 equiv, 0.38 mmol, 53 mg) inCH₃CN (1 mL). The reaction mixture stirred for 12 h at 65° C. The crudereaction mixture was then placed directly on the silica gel plateChromatography (PTLC, SiO₂, 20 cm×20 cm, 1 mm, 1:1 EtOAc-hexane)provided 18 (49 mg, 91 mg theoretical, 54%) as a white solid; LRMS m/z363 (M⁺, C₂₃H₂₉N₃O, requires 363).

EXAMPLE 17N-1-tert-Butoxycarbonyl[3-R-(1′,3′-Dicyclohexyl-ureidocarbonyl)piperidine(19)

A solution of R-Boc-nipecotic acid (6.54 mmol, 1.50 g) and3-aminopyridine (1.1 equiv, 7.20 mmol, 677 mg) in CH₂Cl₂ (25 mL) at 0°C. was treated with DCC (2.0 equiv, 13.08 mmol, 2.70 g) under Ar. Thereaction mixture was allowed to warm to 25° C. and stirred for 12 h. Thereaction mixture was then filtered to remove the urea and the solventswere removed in vacuo. Compound 19 was obtained as a significant sideproduct. Chromatography (SiO₂, 2.5 cm×30.5 cm, 3:1 hexane-EtOAc) gave 19(0.45 g); LRMS m/z 435 (M⁺, C₂₄H₄₁N₃O₄, requires 435).

EXAMPLE 18N-1-Phenethyl[3-R-(1′,3′-Dicyclohexyl-ureidocarbonyl)piperidine (20)

Compound 19 (0.23 mmol, 100 mg) was treated with 20% TFA—CH₂Cl₂ (1 mL)under Ar at 0° C. The reaction mixture stirred for 1 h. The solventswere removed in vacuo and the resulting oil was dried for 3 h undervacuum. The resulting crude amine salt was used directly withoutpurification.

The above compound and phenylacetaldehyde (2.0 equiv, 0.46 mmol, 54 μL)were dissolved in DMF (500 μL) under Ar. After stirring for 1 h at 25°C., the reaction mixture was treated with Na(OAc)₃BH (2.0 equiv, 0.46mmol, 98 mg) and stirred for 12 h at 25° C. The reaction mixture wasquenched with 10% aqueous NaHCO₃ and then extracted with 3×EtOAc (25mL). The organic layer was washed with saturated aqueous NaCl, and driedover MgSO₄. Chromatography (PTLC, SiO₂, 20 cm×20 cm, 1 mm, 9:1EtOAc-CH₃OH) provided 20 (80 mg, 202 mg theoretical, 40%) as a paleyellow solid; LRMS m/z 439 (M⁺, C₂₇H₄₁N₃O₂, requires 439).

EXAMPLE 19N-1-tert-Butoxycarbonyl[3-S-(1′,3′-Dicyclohexyl-ureidocarbonyl)piperidine(21)

A solution of S-Boc-nipecotic acid (6.54 mmol, 1.50 g) and3-aminopyridine (1.1 equiv, 7.20 mmol, 677 mg) in CH₂Cl₂ (25 mL) at 0°C. was treated with DCC (2.0 equiv, 13.08 mmol, 2.70 g) under Ar. Thereaction mixture was allowed to warm to 25° C. and stirred for 12 h. Thereaction mixture was then filtered to remove the urea and the solventswere removed in vacuo. Compound 21 was obtained as a significant sideproduct. Chromatography (SiO₂, 2.5 cm×30.5 cm, 3:1 hexane-EtOAc) gave 21(0.60 g); LRMS m/z 435 (M⁺, C₂₄H₄₁N₃O₄, requires 435).

EXAMPLE 20N-1-Phenethyl[3-S-(1′,3′-Dicyclohexyl-ureidocarbonyl)piperidine (22)

Compound 21 (0.344 mmol, 150 mg) was treated with 50% TFA—CH₂Cl₂ (1 mL)under Ar at 0° C. The reaction mixture stirred for 1 h. The solventswere removed in vacuo and the resulting oil was dried for 3 h undervacuum. The resulting crude amine salt was used directly withoutpurification.

The resulting oil was treated with phenethyl bromide (3.0 equiv, 1.36mmol, 0.140 mL) and K₂CO₃ (2.0 equiv, 0.69 mmol, 95 mg) in CH₃CN (1 mL).The reaction mixture stirred for 12 h at 65° C. and then quenched with 3mL of H₂O. The reaction mixture was then extracted with EtOAc (5 mL).The organic layer was washed with saturated aqueous NaCl, and dried overMgSO₄. Chromatography (PTLC, SiO₂, 20 cm×20 cm, 1 mm, 9:1 (EtOAc-CH₃OH)provided 22 (49 mg, 151 mg theoretical, 32%) as a pale yellow solid;LRMS m/z 439 (M⁺, C₂₇H₄₁N₃O₂, requires 439).

EXAMPLE 21 Synthesis of3(R)-Benzyloxycarbonylamino-piperidine-1-carboxylic Acid Tert-ButylEster

To a solution of piperidine-1,3-dicarboxylic acid 1-tert butyl ester(1.23 g, 5.37 mmol) in THF (40 ml) at 0° C. was added Et₃N (1.90 ml,13.4 mmol), isobutylchloroformate (1.40 ml, 10.74 mmol). After 1 hr ofstirring, sodium azide (6.28 g, 96.7 mmol) in water (21 ml) was addedand the reaction mixture stirred for 30 min. EtOAc (80 ml) was added.The organic layer was separated, washed with water, dried (Na₂SO₄),filtered and concentrated in vacuo. The IR (film) of the crude acylazide had an absorption at 2137(N₃), 1693(C═O) cm⁻¹. The crude residuewas dissolved in dry benzene (100 ml) and heated at 60° C. for 70 min.After removal of benzene, the IR of the crude isocyanate (film) 2994,2865, 2263, 1698, 1422, 1377, 1241, 1169 cm⁻¹; The crude mixture wasdissolved in toluene (10 ml), BnOH (0.7 ml) was added. The mixture washeated at 110° C. for 48 hr (monitored by IR until the absorption at2263cm⁻¹ completely disappeared). After removal of the solvent, theresidue was purified by silica gel chromatography (20% EtOAc in hexane)to give a colorless oil (1.02 g, 57% for three steps). ¹H-NMR (300 MHz,CDCl₃) δ 7.20 (m, 5H), 5.15 (s, 2H), 4.90 (broad s, 1H), 3.75 (broad s,1H), 3.60 (m, 1H), 3.40-3.20 (m, 3H), 2.85 (m, 1H), 1.78-1.50 (m, 2H),1.45 (s, 9H) ppm; IR (film) 3321, 2979, 2858, 1692 (with two shoulders),1540, 1431, 1234, 1154 cm⁻¹; LRMS (calculated for C₁₈H₂₅N₂O₄) 334, found(M-BOC)⁺ 234, (M-BOC+1)⁺ 235.

EXAMPLE 22 Synthesis of 3(R)-amino-piperidine-1-carboxylic AcidTert-Butyl Ester

To a slurry of Pd-C 10%, 170 mg) in MeOH (2 ml) was added a solution ofthe 3(R)-Benzyloxycarbonylamino-piperidine-1-carboxylic acid tert-butylester (1.0 g) in MeOH (60 ml). The mixture was stirred under H₂ (1 atm)for 8 hr. After being filtered through Celite, the filtrate wasevaporated to give a light yellow oil (572 mg, 99%). ¹H-NMR (300 MHz,CDCl₃) δ 4.00-3.78 (m, 2H), 3.80 (m, 2H), 3.60 (m, 1H), 1.90 (m, 1H),1.70 (m, 1H), 1.60-1.40 (m, 12H), 1.30 (m, 1H) ppm; IR (film) 3485,3361, 3292, 2975, 22931, 2862, 1692, 1608, 1548, 1479, 1427, 1367, 1266,1242, 1162 cm⁻¹; LRMS (calculated for C₁₀,H₂₀N₂O₂) 200, found 200.

EXAMPLE 23 Synthesis of3(R)-(2-Nitro-phenylamino)-piperidine-1-carboxylic Acid Tert-Butyl Ester

A mixture of 3(R)-amino-piperidine-1-carboxylic acid tert-butyl ester(137 mg, 0.69 mmol) and 2-nitrofluorobenzene (102 mg, 0.72 mmol), K₂CO₃(100 mg) was heated at 110° C. for 12 hr. The mixture was diluted withCH₂Cl₂, filtered and concentrated. The residue was purified bychromatography (5% EtOAc in hexane) to give a yellow oil (134 mg,61%).¹H-NMR (300 MHz, CDCl₃) δ 8.20 (d, J=8.55 Hz, 1H, NH), 8.10 (d,J=7.20 Hz, 1H), 7.48 (t, 1H), 7.01 (d, J=8.30 Hz, 1H), 6.70 (t, 1H),4.00 (broad, 1H), 3.75 (m, 1H), 3.60 (m, 1H), 3.20 (m, 2H), 2.10 (m,1H), 1.82-1.60 (m, 3H), 1.43 (s, 9H) ppm; IR (film) 3357, 2967, 2935,2858, 1692, 1620, 1572, 15508, 1423, 1372, 1266, 1230, 1154 cm⁻¹; LRMS(calculated for C₆H₂₃N₂O₄) 321, found 321.

EXAMPLE 24 Synthesis of3(R)-(2-Amino-phenylamino)-piperidine-1-carboxylic acid tert-butyl ester

The solution of 3(R)-(2-Nitro-phenylamino)-piperidine-1-carboxylic acidtert-butyl ester (134 mg) and Pd-C (10%, 150 mg) in MeOH (30 ml) wasstirred under H₂ (1 atm) for 24 hrs. After filtration through Celite,the filtrate was concentrated to give colorless oil (96.8 mg, 80%).¹H-NMR (300 MHz, CDCl₃) δ 6.80 (m, 5H), 4.10 (m, 1H), 3.78 (m, 1H), 3.39(m, 3H), 3.15 (m, 1H), 2.95 (m, 1H), 2.08 (m, 1H), 1.80 (m, 1H), 1.60 (m3H), 1.50 (s, 9H) ppm; LRMS (calculated for C₁₆H₂₁N₃O₂-BOC)⁺ 191, found(M-BOC)⁺ 191.

EXAMPLE 25 Synthesis of3(R)-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic AcidTert-Butyl Ester

To a solution of 3(R)-(2-Amino-phenylamino)-piperidine-1-carboxylic acidtert-butyl ester (52 mg, 0.18 mmol), Et₃N (1.0 ml) in CH₂Cl₂ (1 ml) at0° C. was added a solution of phosgene (20% in toluene, 0.1 ml). Afterstirring for 2 hr and evaporation of solvent, the residue was purifiedby chromatography (1%-2% MeOH in CH₂Cl₂) to give a colorless oil (34 mg,60%). ¹H-NMR (300 MHz, CDCl₃) δ 7.80 (d, 1H), 4.20 (m, 3H), 3.50 (m,1H), 2.75 (m, 1H), 2.65 (m, 1H), 2.10 (m, 1H), 1.90 (m, 1H), 1.70 (m,1H), 1.50 (s, 9H) ppm; IR (film) 3067, 2975, 2935, 2858, 1745, 1692,1612, 1487, 1423, 1383, 1347, 1266, 1246, 1158 cm⁻¹; LRMS (calculatedfor C₁₇H₂₃N₃O₃-BOC)⁺ 217, found 217.

EXAMPLE 26 Synthesis of1-(1-Phenethyl-piperidin-3-yl)-1,3-dihydro-benzoimidazol-2-one

3(R)-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acidtert-butyl ester (34 mg) was dissolved in a mixture of TFA—CH₂Cl₂ (1 ml,20%) and stirred for 1 hr. After removal of solvent, the residue wasdried under vacuum for 30 min. The residue was dissolved in 5 ml ofCH₂Cl₂, washed with sat. K₂CO₃, dried over Na₂SO₄, filtered. Evaporationof solvent provided the crude product (20 mg, 86%). LRMS (calculated forC₁₂H₁₅N₃O) 217, found 217; IR (film) 3400-3200 (broad), 2943, 1696,1612, 1487, 1375 cm⁻¹.

The crude product from the above step with phenylacetaldehyde (16 μl,0.14 mmol), Pd-C (10%, 20 mg) in MeOH (1 ml) was stirred under H₂ (1atm) for 14 hr. The catalyst was removed by filtration through Celite.After removal of solvent, the residue was purified by silica gelchromatography (20%EtOAc, 2%-4%MeOH in CH₂Cl₂) to give a colorless oil.¹H-NMR (300 MHz, CDCl₃) δ 9.00 (s, 1H, NH), 7.40-7.00 (m, 9H), 4.50 (m,1H), 3.10 (m, 2H), 2.90-2.60 (m, 4H), 2.30 (m, 1H), 2.18 (m, 1H),2.00-1.78 (m, 4H) ppm; LRMS (calculated for C₂₀H₂₃N₃O+H⁺) 322, found322; IR (film) 3400-3200 (broad), 2947, 1696, 1604, 1491, 1387 cm⁻¹.

EXAMPLE 27 Synthesis of1-(1-Allyl-piperidin-3-ylmethyl)-1-phenyl-3-(4-trifluoromethoxyphenyl-urea(24)

To a solution of N-(1-Boc-piperidin-3-ylmethyl)aniline 1 (460 mg, 1.59mmol) in 5 mL of dry CH₂Cl₂ was added 4-(trifluoromethoxy)-phenylisocyanate (222 μL, 1.59 mmol) at room temperature. After being shakenat room temperature overnight, the reaction mixture was passed throughan aminopropyl NH₂ cartridge and washed with CH₂Cl₂. Removal of CH₂Cl₂afforded 23 (300 mg, 39%). LRMS (M-100)⁺ 393.

Trifluoroacetic acid (0.75 mL) was added dropwise to a solution ofN-(1-Boc-piperidin-3-yl-methyl)-N-phenyl-N′-ethylurea (23) (300 mg, 0.61mmol) in 0.75 mL of dry CH₂Cl₂ at 0° C. (ice-water). The reactionmixture was stirred at room temperature for 30 minutes. TLC showed thereaction was complete. After removal of the solvents, the residue wasdried under vacuum for 3 hrs. The crude product was used for next stepwithout purification.

The crude compound from previous step was dissolved in CH₃CN (1.5 mL)and allyl bromide (106 μL, 1.22 mmol) and potassium carbonate (253 mg,1.83 mmol) were added. The mixture was stirred at room temperature for 2days. The mixture was quenched with 5 mL of water, then extracted withethyl acetate (3×10 mL). The extracts were combined and washed withaqueous NaOH (10%, 5 mL), HCl (5%, 5 mL), and dried over anhydroussodium sulfate. After the solvent was removed, the remaining oilyresidue was purified by preparative thin layer chromatography(CH₂Cl₂/MeOH, 95:5) to afford1-(1-Allyl-piperidin-3-ylmethyl)-1-phenyl-3-(4-trifluoromethoxy-phenyl)-urea24 as colorless oil (40 mg). LRMS 434.

EXAMPLE 281-Phenethyl-3-(1-phenethyl-piperidin-3-ylmethyl)-1,3-dihydro-benzoimidazol-2-one

A solution of 25 (4.84 mmol, 1.11 g), phenethyl bromide (2.0 equiv, 9.68mmol, 1.30 mL) and K₂CO₃ (2.0 equiv, 9.68 mmol, 1.34 g) in CH₃CN (30 mL)was heated at 65° C. for 12 h. The reaction was quenched with 30 mL ofH₂O and then extracted with EtOAc (30 mL). The organic layer was driedwith NaCl (sat) and MgSO₄ (s). Chromatography (SiO₂, 2.5 cm×30.5 cm, 9:1EtOAc-CH₃OH) provided 10 (1.31 g, 1.62 g theoretical, 81%) as a whitesolid, LRMS m/z 335 (M⁺, C₂₁H₂₅N₃O, requires 335). Side product 26 wasalso obtained (0.168 g), LRMS m/z 440 (M⁺, C₂₉H₃₃N₃O, requires 440).

EXAMPLE 293-[2-Oxo-3-(2,2,2-trifluoro-ethyl)-2,3-dihydro-benzoimidazol-1-ylmethyl]-piperidine-1-carboxylicAcid Benzyl Ester

A solution of 9 (0.274 mmol, 100 mg), 2-iodo-trifluoroethane (1.5 equiv,0.411 mmol, 41 μL) and NaH (1.5 equiv, 0.411 mmol, 17 mg) in DMF (500μL) was stirred at 25° C. for 12 h. The reaction was quenched with 5 mLof saturated aqueous NaHCO₃ and then extracted with EtOAc (5 mL). Theorganic layer was dried with NaCl (sat) and NA₂SO₄ (s). Chromatography(PTLC, SiO₂, 20 cm×20 cm, 1 mm, 1:1 EtOAc-hexane) provided 27 (37 mg,123 mg theoretical, 30%) as a white solid; LRMS m/z 447 (M⁺,C₂₃H₂₄F₃N₃O₃, requires 447).

EXAMPLE 30 2-(2-Amino-phenylcarbamoyl)-piperazine-14-dicarboxylic acid1-benzyl ester 4-tert-butyl Ester

A solution of 28 (2.99 mmol, 1.09 g) and 1,2-phenylenediamine (1.0equiv, 2.99 mmol, 0.32 g) in CH₂Cl₂ (10 mL) at 0° C. was treated withDCC (1.5 equiv, 4.49 mmol, 0.93 g) under Ar. The reaction mixture wasallowed to warm to 25° C. and stirred for 12 h. The reaction mixture wasthen filtered to remove the urea and the solvents were removed in vacuo.Chromatography (SiO₂, 2.5 cm×30.5 cm, 1:1 hexane-EtOAc) provided theresulting solid 29 (1.02 g, 1.36 g theoretical, 75%) as a yellowishwhite solid: R_(f) 0.45 (SiO₂, 1:1 hexane-EtOAc); LRMS m/z 454 (M⁺,C₂₄H₃₀N₄O₅, requires 454).

EXAMPLE 312-(2-Oxo-2,3-dihydro-benzoimidazol-1-ylmethyl)-piperazine-1,4-dicarboxylicacid 1-benzyl ester 4-tert-butyl Ester

A solution of 29 (1.10 mmol, 500 mg) in THF (1.0 mL) at 0° C. wastreated with 1.0M BH₃-THF (1.5 equiv, 0.46 mmol) under Ar. The reactionmixture was then heated to 80° C. and allowed to stir for 2.5 h. Thereaction mixture was then cooled to 0° C. and quenched with 10% aqueousHCl. The pH was adjusted to 10 with 10% aqueous NaOH and the reactionmixture was extracted with EtOAc (3×25 mL). The organics were dried withNaCl (sat) and MgSO₄ (s). The material was carried directly to the nextstep without further purification.

The above product in THF (5 mL) at 0° C. was treated with phosgene (20%in toluene) (2.0 equiv, 2.20 mmol, 1.1 mL) and triethylamine (2.0 equiv,2.20 mmol, 307 μL) under Ar. The reaction mixture stirred at 0° C. for30 min and was then warmed to 25° C. for 1 h. The reaction mixture wascooled to 0° C. and quenched with saturated aqueous NaHCO₃ and extractedwith EtOAc (3×25 mL). The organics were dried with NaCl (sat) and MgSO₄(s) and solvents were removed in vacuo. Chromatography (SiO₂, 2.5cm×30.5 cm, 2:1 EtOAc-hexane) provided the resulting solid 30 (0.30 g,0.51 g theoretical, 59%) as a white solid: R_(f) 0.36 (SiO₂, 2:1EtOAc-hexane); LRMS m/z 466 (M⁺, C₂₅H₃₀N₄O₅, requires 466).

EXAMPLE 32 Radioligand Binding Assays

Sodium channel, site 2 assays were conducted according to Catterall, W.A. et al. J. Biol. Chem. 1981, 256, 8922. Calcium channel type L(phenylalkylamine site) assays were conducted according to Reynolds, I.J. et al. J. Pharmacol. Exp. Ther. 1986, 237, 731. Results from a numberof these assays are presented in FIG. 1.

EXAMPLE 33 Radioligand Binding Assays, Tissue Assays, and In VivoAnalgesia Experiments

The μ-opioid receptor assays were conducted according to Wang, J. B. etal. FEBS Letters 1994, 338, 217. The κ-opioid receptor assays wereconducted according to Simonin, F. et al. Proc. Natl. Acad. Sci. USA1995, 92(15), 1431. The 6-opioid receptor assays were conductedaccording to Simonin, F. et al. Mol. Pharmacol. 1994, 92(15), 1015. Theμ- and κ-opioid tissue assays were conducted according to Maguire, P. etal. Eur. J Pharmacol. 1992, 213, 219. The tail flick experiments wereconducted according to D'Amour, F. E. and Smith, D. L. J. Pharmacol.Exp. Ther. 1941, 72, 74. Results from a number of these assays arepresented in FIG. 2.

We claim:
 1. A compound represented by structure A:

wherein X represents CH₂; Z represents O, S or NR; R representsindependently for each occurrence H, C₁-C₁₀ alkyl, aryl or monocyclic orbicyclic heteroaryl with 5-12 ring atoms, of which one to three ringatoms are selected independently from the group consisting of S, O, andN; R₁ represents H, C₁-C₁₀ alkyl, alkenyl, alkynyl, aryl, monocyclic orbicyclic heteroaryl with 5-12 ring atoms, of which one to three ringatoms are selected independently from the group consisting of S, O, andN, C₁-C₂₀ aralkyl or monocyclic or bicyclic heteroaralkyl with 5-12 ringatoms, of which one to three ring atoms are selected independently fromthe group consisting of S, O, and N; R₂ represents independently foreach occurrence H, C₁-C₁₀ alky, alkenyl, alkynyl, aryl, C₁-C₂₀ aralkylor alkyl substituted with monocyclic or bicyclic heteroaryl with 5-12ring atoms, of which one to three ring atoms are selected independentlyfrom the group consisting of S, O, and N, or the two instances or R₂taken together represent an aryl or monocyclic or bicyclic heteroarylwith 5-12 ring atoms, of which one to three ring atoms are selectedindependently from the group consisting of S, O, and N, or a —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, or —CH₂CH₂CH₂CH₂—tether between the two nitrogensof the urea moiety; R₃ represents H, C₁-C₁₀ alkyl, C₁-C₁₀ fluoroalkyl,alkenyl, alkynyl, C₁-C₂₀ aralkyl or alkyl substituted with monocyclic orbicyclic heteroaryl with 5-12 ring atoms, of which one to three ringatoms are selected independently from the group consisting of S, O, andN; m is an integer in the range 1 to 4 inclusive; n is equal to 1; p isan integer in the range 1 to 3 inclusive; and the stereochemicalconfiguration at any stereocenter of a compound represented by A may beR, S, or a mixture of these configurations.
 2. The compound of claim 1,wherein Z represents O.
 3. The compound of claim 1, wherein R representsindependently for each occurrence H or C₁-C₁₀ alkyl.
 4. The compound ofclaim 1, wherein p is
 1. 5. The compound of claim 1, wherein Zrepresents O; and R represents independently for each occurrence H orC₁-C₁₀ alkyl.
 6. The compound of claim 1, wherein Z represents O; and pis
 1. 7. The compound of claim 1, wherein Z represents O; R representsindependently for each occurrence H or C₁-C₁₀ alkyl; and p is
 1. 8. Thecompound of claim 1, wherein said compound is a single enantiomer.